RESUMEN
Inflammation is implicated in the etiology of obesity-related diseases. Thromboxane-prostanoid receptor (TPR) is known to play a role in mediating an inflammatory response in a variety of cells. Gut-derived lipopolysaccharide (LPS), a TLR4 agonist, is elevated in obesity. Moreover, free fatty acids (FFAs) are important mediators of obesity-related inflammation. However, the role and mechanisms by which TPR regulates the inflammatory response in human immune cells remain unclear. We sought to determine the link between TPR and obesity and the role/mechanisms by which TPR alters LPS- or stearic acid (SA)-induced inflammatory responses in PBMCs. Cells were pre-treated with agents blocking TPR signaling, followed by treatment with LPS or stearic acid (SA). Our findings showed that TPR mRNA levels are higher in PBMCs from individuals with obesity. Blockade of TPR as well as ROCK, which acts downstream of TPR, attenuated LPS- and/or SA-induced pro-inflammatory responses. On the other hand, TPR activation using its agonist enhanced the pro-inflammatory effects of LPS and/or SA. Of note, the TPR agonist by itself elicits an inflammatory response, which was attenuated by blocking TPR or ROCK. Our data suggest that TPR plays a key role in promoting an inflammatory response in human PBMCs, and this effect is mediated via TLR4 and/or ROCK signaling.
Asunto(s)
Inflamación , Leucocitos Mononucleares , Lipopolisacáridos , Receptores de Tromboxanos , Transducción de Señal , Ácidos Esteáricos , Humanos , Lipopolisacáridos/farmacología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Ácidos Esteáricos/farmacología , Transducción de Señal/efectos de los fármacos , Inflamación/patología , Inflamación/metabolismo , Receptores de Tromboxanos/metabolismo , Receptores de Prostaglandina/metabolismo , Obesidad/metabolismo , Masculino , FemeninoRESUMEN
Impedance aggregometry is an alternative to light transmission aggregometry that allows analysis of platelet function in whole blood samples. We hypothesized (1) impedance aggregometry would produce repeatable results, (2) inhibition of cyclooxygenase with aspirin would attenuate aggregation responses to collagen and abolish the aggregation response to arachidonic acid (AA), and (3) thromboxane receptor antagonism (terutroban) would attenuate the aggregation response to AA. Venous blood was obtained from 11 participants three times separated by at least 2 weeks. One sample followed 7-day-aspirin intervention (81 mg once daily; ASA), the others no intervention (control). Aggregation was induced using 1 µg/mL collagen ([col 1]), 5 µg/mL collagen ([col 5]), and 50 mM AA via impedance aggregometry to determine total aggregation (AUC) analyzed for intra-test repeatability, inter-test repeatability, intervention (ASA or control), and incubation (saline or terutroban). [col 1] showed high intra-test (p ≤ 0.03 visit 1 and 2) and inter-test repeatability (p < 0.01). [col 5] and AA showed intra- ([col 5] p < 0.01 visit 1 and 2; AA p < 0.001 visit 1 and 2) but not inter-test repeatability ([col 5] p = 0.48; AA p = 0.06). ASA attenuated AUC responses to [col 1] (p < 0.01), [col 5] (p = 0.03), and AA (p < 0.01). Terutroban attenuated AUC in response to AA (p < 0.01). [col 1] shows sufficient repeatability for longitudinal investigations of platelet function. [col 5] and AA may be used to investigate mechanisms of platelet function and metabolism at a single time point.
Asunto(s)
Aspirina , Inhibidores de la Ciclooxigenasa , Impedancia Eléctrica , Agregación Plaquetaria , Pruebas de Función Plaquetaria , Propionatos , Receptores de Tromboxanos , Humanos , Agregación Plaquetaria/efectos de los fármacos , Masculino , Proyectos Piloto , Femenino , Inhibidores de la Ciclooxigenasa/farmacología , Aspirina/farmacología , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/metabolismo , Adulto , Pruebas de Función Plaquetaria/métodos , Propionatos/farmacología , Naftalenos/farmacología , Ácido Araquidónico/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/farmacología , Colágeno/farmacologíaRESUMEN
BACKGROUND: Prostaglandin I2 synthesized by endothelial COX (cyclooxygenase) evokes potent vasodilation in some blood vessels but is paradoxically responsible for endothelium-dependent constriction (EDC) in others. Prostaglandin I2 production and EDC may be enhanced in diseases such as hypertension. However, how PGIS (prostaglandin I2 synthase) deficiency affects EDC and how this is implicated in the consequent cardiovascular pathologies remain largely unknown. METHODS: Experiments were performed with wild-type, Pgis knockout (Pgis-/-) and Pgis/thromboxane-prostanoid receptor gene (Tp) double knockout (Pgis-/-Tp-/-) mice and Pgis-/- mice transplanted with unfractionated wild-type or Cox-1-/- bone marrow cells, as well as human umbilical arteries. COX-derived prostanoids were measured by high-performance liquid chromatography-mass spectrometry. Vasomotor responses of distinct types of arteries were assessed by isometric force measurement. Parameters of hypertension, vascular remodeling, and cardiac hypertrophy in mice at different ages were monitored. RESULTS: PGF2α, PGE2, and a trace amount of PGD2, but not thromboxane A2 (TxA2), were produced in response to acetylcholine in Pgis-/- or PGIS-inhibited arteries. PGIS deficiency resulted in exacerbation or occurrence of EDC ex vivo and in vivo. Endothelium-dependent hyperpolarization was unchanged, but phosphorylation levels of eNOS (endothelial nitric oxide synthase) at Ser1177 and Thr495 were altered and NO production and the NO-dependent relaxation evoked by acetylcholine were remarkably reduced in Pgis-/- aortas. Pgis-/- mice developed high blood pressure and vascular remodeling at 16 to 17 weeks and subsequently cardiac hypertrophy at 24 to 26 weeks. Meanwhile, blood pressure and cardiac parameters remained normal at 8 to 10 weeks. Additional ablation of TP (TxA2 receptor) not only restrained EDC and the downregulation of NO signaling in Pgis-/- mice but also ameliorated the cardiovascular abnormalities. Stimulation of Pgis-/- vessels with acetylcholine in the presence of platelets led to increased TxA2 generation. COX-1 disruption in bone marrow-derived cells failed to affect the development of high blood pressure and vascular remodeling in Pgis-/- mice though it largely suppressed the increase of plasma TxB2 (TxA2 metabolite) level. CONCLUSIONS: Our study demonstrates that the non-TxA2 prostanoids/TP axis plays an essential role in mediating the augmentation of EDC and cardiovascular disorders when PGIS is deficient, suggesting TP as a promising therapeutic target in diseases associated with PGIS insufficiency.
Asunto(s)
Endotelio Vascular , Oxidorreductasas Intramoleculares , Ratones Endogámicos C57BL , Ratones Noqueados , Prostaglandinas , Vasoconstricción , Animales , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/deficiencia , Oxidorreductasas Intramoleculares/metabolismo , Ratones , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Prostaglandinas/metabolismo , Humanos , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/deficiencia , Tromboxano A2/metabolismo , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Enfermedades Cardiovasculares/etiología , Masculino , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxanos/genética , Vasodilatación , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Remodelación Vascular , Transducción de Señal , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismoRESUMEN
We examined the inhibitory effects of α-linolenic acid (ALA) on the contractions of pig coronary arteries. ALA concentration-dependently inhibited the contractions elicited by U46619 and prostaglandin F2α without affecting those elicited by 80 mM KCl, histamine, acetylcholine, and serotonin. ALA rightward shifted the concentration-response curve of U46619, and Schild plot analysis revealed that ALA competitively antagonized U46619. Furthermore, ALA inhibited the increase in intracellular Ca2+ concentration caused by TP receptor stimulation but not that caused by FP receptor stimulation. These results suggest that ALA behaves as a selective antagonist of TP receptors in coronary arteries.
Asunto(s)
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Calcio , Vasos Coronarios , Receptores de Tromboxanos , Ácido alfa-Linolénico , Animales , Vasos Coronarios/efectos de los fármacos , Ácido alfa-Linolénico/farmacología , Porcinos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Calcio/metabolismo , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Dinoprost/farmacología , Contracción Muscular/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. EXPERIMENTAL APPROACH: Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. KEY RESULTS: BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. CONCLUSION AND IMPLICATIONS: F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.
Asunto(s)
Receptores de Tromboxanos , Tromboxano A2 , Tromboxano A2/metabolismo , Humanos , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células HEK293 , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Receptor Cross-Talk/efectos de los fármacosRESUMEN
Excessive mitochondrial fission in podocytes serves as a central hub for the pathogenesis of diabetic nephropathy (DN), and the thromboxane/prostaglandin receptor (TP receptor) plays a potential role in DN. However, regulation of the TP receptor during mitochondrial dynamics disorder in podocytes remains unknown. Here, we firstly reported novel mechanistic details of TP receptor effects on mitochondrial dynamics in podocytes under diabetic conditions. Expression of the TP receptor was significantly upregulated in podocytes under diabetic conditions both in vivo and in vitro. S18886 attenuated podocyte mitochondrial fission, glomerular injury and renal dysfunction in diabetic mice. Furthermore, inhibition of the TP receptor by both genetic and pharmacological methods dramatically reduced mitochondrial fission and attenuated podocyte injury induced by high glucose through regulating dynamin-related protein 1 (Drp1) phosphorylation and its subsequent translocation to mitochondria. In contrast, TP receptor overexpression and application of TP receptor agonist U46619 in these podocytes showed the opposite effect on mitochondrial fission and podocyte injury. Furthermore, treatment with Y27632, an inhibitor of Rho-associated kinase1 (ROCK1), significantly blunted more fragmented mitochondria and reduced podocyte injuries in podocytes with TP receptor overexpression or after U46619 treatment. Finally, pharmacological inhibition of Drp1 alleviated excessive mitochondrial fragmentation and podocyte damage in TP receptor overexpressing podocytes. Our data suggests that increased expression of the TP receptor can occur in a human cultured podocyte cell line and in podocytes derived from streptozotocin (STZ)-induced diabetic mice, which contributes to mitochondrial excessive fission and podocyte injury via ROCK1-Drp1 signaling.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Enfermedades Mitocondriales , Podocitos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/uso terapéutico , Animales , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/patología , Dinaminas/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Ratones , Enfermedades Mitocondriales/metabolismo , Dinámicas Mitocondriales , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , Prostaglandinas/uso terapéutico , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/uso terapéutico , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxanos/uso terapéutico , Estreptozocina , Tromboxanos/metabolismo , Tromboxanos/farmacología , Tromboxanos/uso terapéutico , Quinasas Asociadas a rho/metabolismoRESUMEN
Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática , Receptores de Tromboxanos , Animales , Bleomicina/farmacología , F2-Isoprostanos/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Prostaglandinas/metabolismo , Receptores de Tromboxanos/metabolismo , Tromboxanos/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT-qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3 >> FP > EP1 . The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L-798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl-induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage-dependent Ca2+ channel (VDCC) inhibitor, by 40%-85%. Although LA did not suppress high KCl-induced contractions, it suppressed U46619-induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619-induced Ca2+ increases in TP receptor-expressing cells. In contrast, LA inhibited U46619-induced contractions in the presence of verapamil, which was also suppressed by SKF-96365 (a store-operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid-induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA-induced inhibition of U46619-induced contractions.
Asunto(s)
Ácidos Docosahexaenoicos , Ácido Eicosapentaenoico , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Canales de Calcio Tipo L/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacología , Fundus Gástrico/metabolismo , Cobayas , Músculo Liso , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , ARN Mensajero/metabolismo , Receptores de Tromboxanos/metabolismo , Verapamilo/metabolismo , Verapamilo/farmacologíaRESUMEN
BACKGROUND: Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. METHODS: Human endothelial cell senescence was assessed by senescence-associated ß-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. RESULTS: By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2-COX-2-TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. CONCLUSIONS: These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.
Asunto(s)
Ciclooxigenasa 2 , Dipeptidil Peptidasa 4 , Inflamasomas , Biomarcadores/metabolismo , Senescencia Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Células Endoteliales/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamasomas/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores de Tromboxanos/genética , Receptores de Tromboxanos/metabolismoRESUMEN
BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.
Asunto(s)
Células Endoteliales , Receptores de Tromboxanos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/farmacología , Células Endoteliales/metabolismo , Retroalimentación , Homeostasis , Humanos , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Tromboxano A2/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacologíaRESUMEN
Airborne fine particulate matter (PM2.5) is a major cardiovascular disease environmental risk factor. However, the underlying mechanism of action is not fully understood. Thromboxane is widely known as an important vasoconstrictor substance that binds to G-protein-coupled receptors (GPCR) in arteries and is involved in various cardiovascular diseases. This study examined the effect of PM2.5 on thromboxane A2 receptor (TP) in the mesenteric arteries and the underlying intracellular signal mechanisms (by focusing on the mitogen-activated protein kinase (MAPK) pathway). Rat mesenteric artery segments were exposed to PM2.5 in the presence of MAPK pathway inhibitors. The contractile reactivity of mesenteric arteries was analyzed using wire myography. The mRNA and protein expression of TP receptor and MAPK pathway molecules were detected by real-time PCR and Western blot. Mesenteric artery receptor localization was assessed by immunohistochemistry. The results showed that TP receptor-mediated maximum contraction response was achieved after exposing arteries to 1.0 µg/mL PM2.5 for 16 h (Emax: 228 ± 16% of K+). Moreover, inhibitor U0126 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) depressed the increased TP receptor-mediated contractile responses (reduced rage were 17.9 ~ 59.6%). These inhibitors also decreased the increased mRNA expression and protein of the TP receptor induced by PM2.5 (reduced by more than 50% and 46%, respectively). The immunoreactivity of increased TP receptor expression was primarily localized in the cytoplasm. In addition, phosphorylation quantitative analysis showed that in the presence of MAPK inhibitors, the PM2.5-induced phosphorylation of ERK1/2, p38, and JNK protein increased by more than 30.0 ~ 130.3%. These results suggest that PM2.5 upregulates the TP receptor of rat mesenteric arteries through activation of the ERK1/2, p38, and JNK MAPK pathways.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Receptores de Tromboxanos , Animales , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Material Particulado/química , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Tromboxanos/metabolismo , Tromboxanos , Regulación hacia ArribaRESUMEN
12/15-LO (12/15-lipoxygenase), encoded by Alox15 gene, metabolizes arachidonic acid to 12(S)-HETE (12-hydroxyeicosatetraenoic acid). Macrophages are the major source of 12/15-LO among immune cells, and 12/15-LO plays a crucial role in development of hypertension. Global Alox15- or macrophage-deficient mice are resistant to Ang II (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to Ang II-mediated arterial constriction and thus to development of Ang II-induced hypertension. Ang II constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nmol/L) alone was without effect; however, it significantly enhanced Ang II-induced constriction. The presence of wild-type macrophages also enhanced the Ang II-induced constriction, while Alox15-/- macrophages did not. Using this model, pretreatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-mediated, Ang II receptor-2-mediated and superoxide-mediated enhancing effect of 12(S)-HETE on Ang II constrictions. The role of superoxide was confirmed using aortas from p47phox-/- mice where 12(S)-HETE failed to enhance constriction to Ang II. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE or Ang II increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of Ang II constriction. Both Ang II-induced hypertension and the enhancing effect of 12(S)-HETE on Ang II contractions were eliminated by a BLT2 (leukotriene B4 receptor-2) antagonist. These results outline a mechanism where the macrophage 12/15-LO pathway enhances the action of Ang II. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of Ang II in part by promoting endothelial synthesis of a superoxide-derived TP agonist.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Angiotensina II/farmacología , Aorta/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Receptores de Leucotrieno B4/metabolismo , Receptores de Tromboxanos/metabolismo , Animales , Aorta/metabolismo , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Noqueados , Superóxidos/metabolismoRESUMEN
AIMS: We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. MAIN METHODS: Changes in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. KEY FINDINGS: U46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). SIGNIFICANCE: Prostanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.
Asunto(s)
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Receptores de Tromboxanos/metabolismo , Vejiga Urinaria/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/uso terapéutico , Animales , Cobayas , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Técnicas de Cultivo de Órganos , Receptores de Tromboxanos/agonistas , Vejiga Urinaria/efectos de los fármacos , Enfermedades de la Vejiga Urinaria/tratamiento farmacológico , Enfermedades de la Vejiga Urinaria/metabolismo , Vasoconstrictores/farmacología , Vasoconstrictores/uso terapéuticoRESUMEN
AIMS: Thromboxane (TxA2) is synthesized from arachidonic acid (AA) via thromboxane synthase (TxS) enzyme and induces vasoconstriction via TP receptor. Our aim is to compare the effects of aspirin, TxS inhibitor and TP receptor antagonist on vascular reactivity of bypass grafts (saphenous vein and internal mammary artery). MAIN METHODS: Using isolated organ bath, saphenous vein and internal mammary artery preparations were incubated with TP receptor antagonist, TxS inhibitor, aspirin, IP or EP4 receptor antagonist. Then prostaglandin (PG)E2, PGF2α, phenylephrine and AA were administered in concentration-dependent manner. The expression of prostanoid receptor and PGI2 synthase (PGIS) enzyme was determined by Western Blot. KEY FINDINGS: TP receptor antagonist inhibited the contraction induced by PGE2, PGF2α, and AA but not that induced by phenylephrine in both types of vessels. Aspirin increased phenylephrine-induced contraction only in internal mammary artery and decreased AA-induced contraction in saphenous vein. TxS inhibitor decreased both PGE2 and AA-induced contraction in both types of vessels. This decrease was reversed by co-incubation of TxS inhibitor and IP/EP4 receptor antagonists. The expressions of EP3 receptor and PGIS enzyme were greater in internal mammary artery compared to saphenous vein while IP and TP receptors expressed at similar levels. SIGNIFICANCE: TP receptor antagonist and TxS inhibitor are more effective to reduce contraction induced by different spasmogens in comparison to aspirin. Our results suggest that TP receptor antagonist and TxS inhibitor might have an advantage over aspirin due to their preventive effect on increased vascular reactivity observed in post-operative period of coronary artery bypass grafting.
Asunto(s)
Arterias Mamarias/efectos de los fármacos , Vena Safena/efectos de los fármacos , Ácido Araquidónico/metabolismo , Aspirina/farmacología , Benzofuranos/farmacología , Carbazoles/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Arterias Mamarias/metabolismo , Músculo Liso Vascular/metabolismo , Fenilefrina/farmacología , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/efectos de los fármacos , Receptores de Tromboxanos/metabolismo , Vena Safena/metabolismo , Sulfonamidas/farmacología , Tromboxano A2/farmacología , Tromboxano-A Sintasa/antagonistas & inhibidores , Tromboxano-A Sintasa/efectos de los fármacos , Tromboxano-A Sintasa/metabolismo , Tromboxanos/antagonistas & inhibidores , Tromboxanos/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
Thromboxane receptor (TP) mediates nasal obstruction, a typical symptom of allergic rhinitis. Since it has been reported that several types of eicosanoids, such as non-enzymatic oxidation product of arachidonic acid isoprostane, act as a TP ligand, there is a possibility that some other eicosanoids contribute to the TP-mediated nasal obstruction. The aim of this study is to investigate the mechanisms of TP-mediated nasal obstruction. Intranasal challenges of ovalbumin (OVA) induced nasal obstruction in mice. Pharmacological blockade of TP receptor but not thromboxane A2 synthase inhibited OVA-induced nasal obstruction. Simultaneous analysis of eicosanoids in nasal lavage fluid and the responses in trans-endothelial resistance suggested that 8-iso-prostaglandin E2 (PGE2 ) can be a candidate for TP ligand. Intranasal challenge of 8-iso-PGE2 induced vascular hyperpermeability and nasal obstruction in TP receptor-dependent manner. Wholemount immunostaining of nasal septum mucosa revealed that 8-iso-PGE2 increased plasma leakage accompanied by distention of venous sinusoids. This study shows that 8-iso-PGE2 is a contributor in TP-mediated nasal obstruction in mice.
Asunto(s)
Dinoprostona/análogos & derivados , Modelos Animales de Enfermedad , Isoprostanos/farmacología , Obstrucción Nasal/inducido químicamente , Obstrucción Nasal/complicaciones , Receptores de Tromboxanos/metabolismo , Rinitis Alérgica/complicaciones , Rinitis Alérgica/metabolismo , Administración Intranasal , Animales , Permeabilidad Capilar/efectos de los fármacos , Dinoprostona/administración & dosificación , Dinoprostona/farmacología , Femenino , Isoprostanos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/efectos de los fármacosRESUMEN
Hypertension is one of the most common complications of chronic kidney disease (CKD). Some research has indicated that changes in large artery function especially caused by thromboxane A2 (TXA2) may be a novel factor acting to induce hypertension in CKD. We studied the 5/6 nephrectomy rat model and measured serum levels of creatinine (Cr), calcium (Ca), phosphorus (P), TXA2-stable metabolites (thromboxane B2, TXB2), and caudal artery pressure after nephrectomy. The tension variations in thoracic aortas were measured after stimulating by vasoconstrictor/vasodilator using the cumulative concentration administration method and then tested the expression of TXA2 receptors in the thoracic aortas through western blots. The CKD rats developed uremia, electrolyte imbalancesï¼and hypertension. They also exhibited a significant increase in TXB2 concentration. The aortic rings of CKD rats showed an increased contraction response to U46619 (a TXA2 analogue) and the expression of TXA2 receptors also enhanced. In the meanwhile, the diastolic function decreased in the CKD group. Our results demonstrate that the impairment of artery contractile function caused by the increase of TXA2 receptors on the wall of aortic rings may be involved in hypertension in CKD rats.
Asunto(s)
Hipertensión/patología , Receptores de Tromboxanos/metabolismo , Insuficiencia Renal Crónica/complicaciones , Tromboxano A2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hipertensión/etiología , Hipertensión/fisiopatología , Masculino , Ratas , Receptores de Tromboxanos/análisis , Tromboxano A2/análisis , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacologíaRESUMEN
Although commonly thought to produce prostacyclin (prostaglandin I2 ; PGI2 ) that evokes vasodilatation and protects vessels from the development of diseases, the endothelial cyclooxygenase (COX)-mediated metabolism has also been found to release substance(s) called endothelium-derived contracting factor(s) (EDCF) that causes endothelium-dependent contraction and implicates in endothelial dysfunction of disease conditions. Various mechanisms have been proposed for the process; however, the major endothelial COX metabolite PGI2 , which has been classically considered to activate the I prostanoid receptor (IP) that mediates vasodilatation and opposes the effects of thromboxane (Tx) A2 produced by COX in platelets, emerges as a major EDCF in health and disease conditions. Our recent studies from genetically altered mice further suggest that vasomotor reactions to PGI2 are collectively modulated by IP, the vasoconstrictor Tx-prostanoid receptor (TP; the prototype receptor of TxA2 ) and E prostanoid receptor-3 (EP3; a vasoconstrictor receptor of PGE2 ) although with differences in potency and efficacy; a contraction to PGI2 reflects activities of TP and/or EP3 outweighing that of the concurrently activated IP. Here, we discuss the history of endothelium-dependent contraction, evidences that support the above hypothesis, proposed mechanisms for the varied reactions to endothelial PGI2 synthesis as well as the relation of its dilator activity to the effect of another NO-independent vasodilator mechanism, the endothelium-derived hyperpolarizing factor. Also, we address the possible pathological and therapeutic implications as well as questions remaining to be resolved or limitations of our above findings obtained from genetically altered mouse models.
Asunto(s)
Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Vasoconstricción/fisiología , Animales , Endotelio Vascular/efectos de los fármacos , Humanos , Ratones , Prostaglandinas/metabolismo , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxanos/metabolismo , Tromboxanos/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/metabolismoRESUMEN
BACKGROUND: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. METHODS: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. RESULTS: Thromboxane A2 (TXA2)-agonist induced reduced vasoconstriction, testosterone (T) and 17-ß-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. CONCLUSIONS: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA2 and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.
Asunto(s)
Arteriolas/patología , Enfermedad de la Arteria Coronaria/patología , Estradiol/farmacología , Testosterona/farmacología , Tromboxano A2/farmacología , Deficiencia de Vitamina D/complicaciones , Andrógenos/farmacología , Animales , Arteriolas/metabolismo , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/metabolismo , Modelos Animales de Enfermedad , Estrógenos/farmacología , Masculino , Ratas , Ratas Wistar , Receptores de Tromboxanos/metabolismo , Vasoconstricción , Deficiencia de Vitamina D/metabolismo , Deficiencia de Vitamina D/patologíaRESUMEN
Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A2 in their pathophysiology remains unclear. We investigated the systemic TXA2 biosynthesis in vivo and gene expression of its receptor TP in 22 essential hypertension patients and a mouse model of salt-sensitive hypertension. The contribution of platelet TXA2 biosynthesis on enhanced blood pressure (BP) and overload-induced cardiac fibrosis was explored in mice by treating with low-dose Aspirin, resulting in selective inhibition of platelet cyclooxygenase (COX)-1-dependent TXA2 generation. In essential hypertensive patients, systemic biosynthesis of TXA2 [assessed by measuring its urinary metabolites (TXM) reflecting predominant platelet source] was enhanced together with higher gene expression of circulating leukocyte TP and TGF-ß, vs. normotensive controls. Similarly, in hypertensive mice with prostacyclin (PGI2) receptor (IP) deletion (IPKO) fed with a high-salt diet, enhanced urinary TXM, and left ventricular TP overexpression were detected vs. normotensive wildtype (WT) mice. Increased cardiac collagen deposition and profibrotic gene expression (including TGF-ß) was found. Low-dose Aspirin administration caused a selective inhibition of platelet TXA2 biosynthesis and mitigated enhanced blood pressure, cardiac fibrosis, and left ventricular profibrotic gene expression in IPKO but not WT mice. Moreover, the number of myofibroblasts and extravasated platelets in the heart was reduced. In cocultures of human platelets and myofibroblasts, platelet TXA2 induced profibrotic gene expression, including TGF-ß1. In conclusion, our results support tailoring low-dose Aspirin treatment in hypertensive patients with unconstrained TXA2/TP pathway to reduce blood pressure and prevent early cardiac fibrosis.
Asunto(s)
Antifibróticos/farmacología , Antihipertensivos/farmacología , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Cardiomiopatías/prevención & control , Hipertensión Esencial/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Tromboxano A2/sangre , Adulto , Animales , Biomarcadores/sangre , Plaquetas/metabolismo , Cardiomiopatías/sangre , Cardiomiopatías/etiología , Cardiomiopatías/patología , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión Esencial/sangre , Hipertensión Esencial/complicaciones , Hipertensión Esencial/fisiopatología , Femenino , Fibrosis , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Receptores de Epoprostenol/genética , Receptores de Epoprostenol/metabolismo , Receptores de Tromboxanos/metabolismoRESUMEN
This study was to determine how endothelium-dependent contractions (EDCs) change in iliac arteries of Wistar-Kyoto (WKYs) and spontaneously hypertensive rats (SHRs) during the transition from adolescence to adulthood and the underlying mechanism(s). We also aimed to elucidate effects of L-798106, an EP3 receptor antagonist, on EDCs and the blood pressure increase in adolescent SHRs. Blood vessels were isolated for functional and biochemical analyses. EDCs were comparable in adolescent iliac arteries of both strains, and contractions to ACh, prostacyclin (PGI2), the EP3 receptor agonist sulprostone and the TP receptor agonist U46619 in adult vessels were less prominent compared with those in the adolescents, while the attenuation of vasoconstrictions to ACh, PGI2 or U46619 with age was to a lesser extent in SHRs. PGI2 production was decreased to a similar level in adult arteries. TP and EP3 expressions were downregulated in adult vessels, whereas the extent of TP downregulation was less in SHRs. L-798106 partially suppressed the vasoconstrictions to U46619 and attenuated EDCs to a greater extent than SQ29548, and administration of L-798106 blunted the blood pressure increase with age in prehypertensive SHRs. These results demonstrate the comparable EDCs in iliac arteries of the adolescents are decreased in the adults, but relatively larger EDCs in adult SHRs can be a reflection of differential downregulation of TP and EP3 receptors during the transition from adolescence to adulthood. Also, our data suggest that blockade of both TP and EP3 receptors starting from the prehypertensive stage suppresses EDCs and the development of hypertension in SHRs.